RESUMEN
Low success rates during drug development are due, in part, to the difficulty of defining drug mechanism-of-action and molecular markers of therapeutic activity. Here, we integrated 199,219 drug sensitivity measurements for 397 unique anti-cancer drugs with genome-wide CRISPR loss-of-function screens in 484 cell lines to systematically investigate cellular drug mechanism-of-action. We observed an enrichment for positive associations between the profile of drug sensitivity and knockout of a drug's nominal target, and by leveraging protein-protein networks, we identified pathways underpinning drug sensitivity. This revealed an unappreciated positive association between mitochondrial E3 ubiquitin-protein ligase MARCH5 dependency and sensitivity to MCL1 inhibitors in breast cancer cell lines. We also estimated drug on-target and off-target activity, informing on specificity, potency and toxicity. Linking drug and gene dependency together with genomic data sets uncovered contexts in which molecular networks when perturbed mediate cancer cell loss-of-fitness and thereby provide independent and orthogonal evidence of biomarkers for drug development. This study illustrates how integrating cell line drug sensitivity with CRISPR loss-of-function screens can elucidate mechanism-of-action to advance drug development.
Asunto(s)
Antineoplásicos/farmacología , Sistemas CRISPR-Cas , Desarrollo de Medicamentos/métodos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Redes Reguladoras de Genes/efectos de los fármacos , Aptitud Genética/efectos de los fármacos , Mapas de Interacción de Proteínas/efectos de los fármacos , Antineoplásicos/toxicidad , Biomarcadores/metabolismo , Línea Celular Tumoral , Técnicas de Inactivación de Genes , Redes Reguladoras de Genes/genética , Aptitud Genética/genética , Genómica , Humanos , Modelos Lineales , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Preparaciones Farmacéuticas/metabolismo , Programas Informáticos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Micro-RNAs (miRNAs) can be specifically expressed in forensically relevant body fluids such as blood or saliva. The aim of the study was to develop a simultaneous extraction and analysis protocol that allows for the acquisition of a DNA profile and the identity of the body fluid using a single process. DNA and micro-RNA were extracted from blood and saliva before undergoing a cDNA synthesis step by using stem-loop reverse transcription PCR. The resulting extracts containing DNA and cDNA synthesized from body fluid-specific miRNA markers then underwent standard STR analysis using a modified ABI AmpFâSTR(®) NGM SElect™ kit. In all samples, a full DNA profile was obtained along with additional peaks corresponding to the miRNA marker targeted. In all cases, blood samples profiled exhibited a peak indicating the presence of the blood-specific miRNA marker and the saliva sample profiled exhibited a peak indicating the presence of the saliva-specific miRNA marker.